Since its discovery, the proteasome has generally been viewed as a passive player in the selection of substrates for degradation. This notion has recently been challenged by studies, many from this laboratory, showing that proteasomes rapidly edit ubiquitin chains and that these processes strongly regulate proteasome output. We first examined these issues in yeast, but now propose to focus on mammals, where little is being done. The regulatory systems differ between yeast and mammals, making it especially important that these questions are studied carefully in mammals. For example, whereas yeast has a single enzyme that trims chains on the proteasome, Ubp6, mammals have two: Usp14 (the Ubp6 ortholog) and Uch37. Also, our studies indicate that the influence of chain trimming on degradation rates is more dramatic in mammals. We have developed a specific, small-molecule inhibitor of Usp14. Using this inhibitor, both in vivo and in cells, we discovered an unexpectedly powerful role of chain trimming in regulating degradation. This compound is unique in providing, for the first time, a way to enhance output of the ubiquitin-proteasome pathway. Remarkably, it induces faster degradation of multiple proteotoxic proteins as well as oxidized proteins, and provides resistance to proteotoxic stress. Possible eventual therapeutic applications include, in principle, tens of known diseases in which toxic proteins are expressed or protein degradation is deficient. A screen has also been completed for Uch37, covering 130,000 compounds, and a highly selective inhibitor is in hand. Though Usp14 and Uch37 both trim chains, they seem to regulate the proteasome in distinct ways, for unknown reasons. To understand this, we will first carry out in vitro and model substrate studies testing whether the topological linkage specificity of substrate-bound ubiquitin chains provides an underlying code that could explain the substrate specificities of Usp14 and Uch37 and their effects on degradation rates. Similar model substrate work will assess whether a protein's susceptibility to unfolding by the proteasome in linked to chain trimming. What features of the substrate determine susceptibility to editing factors will be analyzed further using endogenous substrates? We will use state of the art mass spectrometry approaches to determine, on a broad, if not global, scale, which substrates in the cell are regulated via proteasomal chain editing, using the inhibitors as well and gene knockouts in mice. Relevant properties of endogenous substrates, such as the type of ubiquitin chain they carry and their ubiquitin receptor dependence, will be characterized. A final component of the editing system is Hul5, a conserved, proteasome-associated ubiquitin ligase. We have shown Hul5 extends proteasome-bound ubiquitin chains and is thus important for chain editing in yeast, where it antagonizes Ubp6. Thus far Hul5 has essentially not been studied in mammals. The results will provide important new insights into proteasome function and might have relevance to the development of novel therapeutics.